Chondroitin synthase

ABSTRACT

A vector of the present invention has DNA encoding a protein or a product having the same effect as the protein, the protein containing an amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO:2. Expression of the DNA gives human chondroitin synthase. By using human chondroitin synthase, it is possible to produce a saccharide chain having a repeating disaccharide unit of chondroitin. The DNA or part thereof may be used as a probe for hybridization for the human chondroitin synthase.

TECHNICAL FIELD

The present invention relates to (a) a vector having DNA encodingchondroitin synthase, (b) a method of producing chondroitin synthase,(c) a method of producing a saccharide chain having a repeatingdisaccharide unit of chondroitin, and (d) a probe for hybridization ofchondroitin synthase.

BACKGROUND ART

Chondroitin sulfate, which is a kind of glycosaminoglycan (GAG), existsas a proteoglycan on cell surfaces and in an extra cellular matrix.Chondroitin sulfate draws attention because Chondroitin sulfate plays animportant role in neural network formation in the developing mammalianbrain (Arch. Biochem. Biophys. 374, 24-34 (2000); Trends Glycosci,Glycotechnol. 12, 321-349 (2000)).

Chondroitin sulfate has a straight-chained polymer structure having arepeating disaccharide unit having a glucuronic acid residue (GlcUA) andan N-acetylgalactosamine residue (GalNAc). A serine residue in a coreprotein is covalent-bonded with chondroitin sulfate via 4-saccharidestructure (GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1) peculiar thereto(Glycoproteins, ed. Gottschalk, A. (Elsevier Science, New York), pp.491-517 (1972); The Biochemistry of Glycoproteins and Proteoglycans, ed.Lennarz, W. J. (Plenum, New York), pp. 267-371 (1980)).

GAG is biosynthesized by sequentially transferring saccharides fromUDP-sugar to a non-reducing end of a saccharide chain. It was found that(a) purification of bovine serum gave a glycosyltransferase thatinvolves in biosynthesis of a repeating disaccharide unit ofheparin/heparan sulfate, and (b) cDNA cloning revealed that a singleprotein of the glycosyltransferase catalyses both transferase reactionsof N-acetylglucosamine residue (GlcNAc) and GlcUA.

On the other hand, a glycosyltransferase that involved in biosynthesisof the repeating disaccharide unit of chondroitin sulfate has not beencloned yet except the chondroitin synthase derived from a bacterium (J.Biol. Chem. 275, 24124-24129 (2000)). GlcUA transferase II (GlcAT-II)and GalNAc transferase II (GalNAcT-II) have been purified from aviancartilage (J. Biol. Chem. 272, 14399-14403 (1997)) and from bovineserum(Eur. J. Biochem. 264, 461-467 (1999)). However, cDNA cloning of thoseenzymes has not been performed yet because it is difficult to purifythose enzymes to form homogeneity.

An object of the present invention is to provide (a) a vector having DNAencoding human chondroitin synthase, (b) a method of producing humanchondroitin synthase, (c) a method of producing a saccharide chainhaving a repeating disaccharide unit of chondroitin, and (d) a probe forhybridization of human chondroitin synthase.

DISCLOSURE OF INVENTION

By searching through a human cDNA database, inventors of the presentinvention successfully found out a candidate DNA encoding humanchondroitin synthase. The inventors accomplished the present inventionby actually expressing the candidate DNA and confirming that thecandidate DNA encodes finding human chondroitin synthase.

The present invention provides the followings:

-   -   (1) A vector carring one of DNA (a), (b) or (c) the DNA (b)        or (c) encoding a protein having catalytic activities (α) and        (β), excluding a DNA encoding a protein at amino-acid position        #1 to 802 in SEQ. ID. NO: 2):

(a) DNA encoding a protein having an amino acid sequence from amino acidnumbers 47 to 802 in SEQ. ID. NO: 2;

(b) DNA that, in a stringent condition, hybridizes with the DNA (a), theDNA complementary with DNA (a), or DNA having part of a nucleotidesequence of the DNA (a) or the DNA complementary with the DNA (a);

(c) DNA encoding a protein having an amino acid sequence from amino acidnumbers 47 to 802 in SEQ. ID. NO:2, wherein one or several amino acidsin the amino acid sequence are substituted, deleted, inserted, ortranspositioned;

(α) catalytic activity that transferase GalNAc from UDP-GalNAc tochondroitin;

-   -   (where UDP is uridine 5′diphosphate and GalNAc is        N-acetylgalactosamine residue), and

(β) catalytic activity that transferase GlcUA from UDP-GlcUA tochondroitin,

-   -   (where UDP is uridine 5′diphosphate and GlcUA is glucuronic acid        residue).

(2) The vector as set forth (1), wherein:

-   -   the DNA (a) corresponds to nucleotide numbers 633 to 2900 in        SEQ. ID. NO: 1.

(3) The vector as set forth in (1) or (2), wherein:

-   -   the proteins are soluble.

(4) The vector as set forth in any one of (1) to (3), being anexpression vector.

(5) A transformant whose host is transformed by a vector having any oneof DNA (a) to (c), the DNA (b) or (c) encoding a protein havingcatalytic activities (α) and (β):

(a) DNA encoding a protein having an amino acid sequence from amino acidnumbers 47 to 802 in SEQ. ID. NO: 2;

(b) DNA that, in a stringent condition, hybridizes with the DNA (a), theDNA complementary with DNA (a), or DNA having part of a nucleotidesequence of the DNA (a) or the DNA complementary with the DNA (a);

(c) DNA encoding a protein having an amino acid sequence from amino acidnumbers 47 to 802 in SEQ. ID. NO:2, wherein one or several amino acidsin the amino acid sequence are substituted, deleted, inserted, ortranspositioned;

(α) catalytic activity that transferase GalNAc from UDP-GalNAc tochondroitin;

-   -   (where UDP is uridine 5′diphosphate and GalNAc is        N-acetylgalactosamine residue),

(β) catalytic activity that transferase GlcUA from UDP-GlcUA tochondroitin,

-   -   (where UDP is uridine 5′diphosphate and GlcUA is N-glucuronic        acid residue).

(6) The transformant as set forth in (5), wherein:

-   -   the DNA (a) encodes finding from nucleotide numbers 633 to 2900        in SEQ. ID. NO: 1.

(7) The transformant as set forth in (5) or (6), wherein:

-   -   the proteins are soluble.

(8) A method for producing chondroitin synthase, the method comprisingthe steps of:

-   -   growing a transformant set forth in any one of (5) to (7); and    -   obtaining the chondroitin synthase from the trnasformant thus        grown.

(9) A reagent for use in chondroitin synthesis, the reagent having anenzyme protein that has an amino acid sequence including an amino acidsequence (A) or (B) and has catalytic activities (α) and (β):

(A) amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID.NO: 2;

(B) amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID.NO:2, wherein one or several amino acids in the amino acid sequence aresubstituted, deleted, inserted, or transpositioned.

(α) catalytic activity that transferase GalNAc from UDP-GalNAc tochondroitin,

-   -   (where UDP is uridine 5′diphosphate, and GalNAc is        N-acetylgalactosamine residue);

(β) catalytic activity that transferase GlcUA from UDP-GlcUA tochondroitin,

-   -   (where UDP is uridine 5′diphosphate and GlcUA is N-glucuronic        acid residue).

(10) The reagent as set forth in (9), wherein:

-   -   the enzyme protein is soluble.

(11) Method for producing a saccharide chain expressed by Formula (3),the method comprising at least the step of causing a reagent to contactwith GalNAc donor and a saccharide chain expressed by Formula (1), thereagent set forth in (9) or (10):GlcUA-GalNAc-R¹   (1),GalNAc-GlcUA-GalNAc-R¹   (3),(where GlcUA and GalNAc are as defined above, “-” indicates a glycosidelinkage, R¹ is an arbitrary group).

(12) A method for producing a saccharide chain expressed by Formula (4),the method comprising at least the step of causing a reagent to contactwith GlcUA donor and a saccharide chain expressed by Formula (2), thereagent set forth (9) or (10):GalNAc-GlcUA-R²   (2),GlcUA-GalNAc-GlcUA-R²   (4),(where GlcUA, GalNAc, and “-” are as defined above, R² is an arbitrarygroup).

(13) A method for producing a saccharide chain selected from saccharidechains expressed by Formulas (5) and (7) respectively, the methodcomprising at least the step of causing a reagent to contact with GalNAcdonor, GlcUA donor and a saccharide chain expressed by Formula (1), thereagent set forth in claim (9) or (10):GlcUA-GalNAc-R¹   (1),(GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (5),GalNAc-(GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (7),(where n is an integer not less than 1, GlcUA, GalNAc, and “-” are asdefined above, R¹ is an arbitrary group).

(14) A method for producing a saccharide chain selected from saccharidechains expressed by Formulas (6) and (8) respectively, the methodcomprising at least the step of causing a reagent to contact with GalNAcdonor, GlcUA donor and a saccharide chain expressed by Formula (2), thereagent set forth in (9) or (10):GalNAc-GlcUA-R²   (2),(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (6),GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (8),(where n is an integer not less than 1, GlcUA, GalNAc, and “-” are asdefined above, R is an arbitrary group).

(15) A probe for hybridization, the probe containing a nucleotidesequence from nucleotide numbers 495 to 2900 in SEQ. ID. NO: 1, or asequence complementary with part of the nucleotide sequence.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates comparison among a putative amino acid sequence ofhuman chondroitin synthase (Human), and amino acid sequences ofhomologous proteins of C. elegans (T25233) and Drosophila (AE003499).Those putative amino acid sequences were analyzed by using GENETYX-MAC(version 10) computer program. Respectively, the black boxes indicatethat three of them have an identical amino acid, and the gray boxesindicate that two of them have an identical amino acid. The broken linesindicate gaps inserted for attaining highest degree of matching.Surrounded by the rectangular frames are predicted transmembrane domain.A D×D motif that was preserved is indicated by underline. Three sitespredicted N-glycosylation sites are marked with star marks.

FIG. 2 shows a genome structure of the human chondroitin synthasegeneExon regions are indicated by the boxes. The black boxes indicatecoding sequences, whereas the white boxes indicate 5′- and3′-untranslated sequences. The translation initiation codon (ATG) andstop codon (TAA) are shown as well. The black horizontal line indicatesintrons.

FIGS. 3(a) and 3(b) show results of identification of reaction productsfrom human chondroitin synthase reaction. FIG. 3(a): A reaction productof GlcUA transferase collected from a Superdex peptide column wasdigested by chondroitinase AC-II or β-glucuronidase. The reactionproduct (black rectangle) that was not digested, the reaction product(black circle) that was digested by chondroitinase AC-II, and thereaction product that was digested by β-glucuronidase, were applied intothe Superdex peptide column. Radioactivity of elution fractions of each(0.4 ml each) was analyzed. Arrows indicate elution positions ofsaturated disaccharide (1, GlcUAβ1-3GalNAc) or isolated GlcUA (2,[¹⁴C]GlcUA). FIG. 3(b): A reaction product of GalNAc transferasecollected from a Superdex peptide column was digested by chondroitinaseAC-II. The reaction product (black rectangle) that was not digested, orthe reaction product (black circle) that was digested by chondroitinaseAC-II, was applied into the Superdex peptide column. Radioactivity ofelution fractions of each (0.4 ml each) was analyzed. Arrows indicateelution positions of saturated disaccharide (1, GlcUAβ1-3GalNAc), orisolated GalNAc (2, [³H]GalNAc).

FIG. 4 shows a result of Northern blot analysis (a photograph ofgel-electrophoresis) of chondroitin synthase in a human tissue.Hybridization of RNAs derived from various human tissues was carried outby using probes of chondroitin synthase: Lane 1 is brain; Lane 2 isheart; Lane 3 is skeletal muscle; Lane 4 is colon; Lane 5 is thymus;Lane 6 is spleen; Lane 7 is kidney; Lane 8 is liver; Lane 9 is smallintestine; Lane 1 is placenta; Lane 11 is lung, and Lane 12 is leukocytein peripheral blood.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail below with reference to anembodiment of the invention.

(1) Vector of the Invention

A vector of the present invention is a vector carrying one of DNA (a),(b) or (c), excluding a DNA encoding a protein at amino-acid position #1to 802 in SEQ. ID. NO: 2):

(a) DNA encoding a protein having an amino acid sequence from amino acidnumbers 47 to 802 in SEQ. ID. NO: 2;

(b) DNA that, in a stringent condition, hybridizes with the DNA (a), theDNA complementary with DNA (a), or DNA having part of a nucleotidesequence of the DNA (a) or the DNA complementary with the DNA (a);

(c) DNA encoding a protein having an amino acid sequence from amino acidnumbers 47 to 802 in SEQ. ID. NO:2, wherein one or several amino acidsin the amino acid sequence are substituted, deleted, inserted, ortranspositioned;

-   -   the above DNA (b) or (c) encoding a protein having catalytic        activities (α) and (β),

(α) catalytic activity that transferase GalNAc from UDP-GalNAc tochondroitin; and

(β) catalytic activity that transferase GlcUA from UDP-GlcUA tochondroitin.

Note that, the foregoing chondroitin is a polymer made of repeatingdisaccharide units of GlcUA and GalNAc. The chondroitin includes onewhose non-reducing end is GlcUA and one whose non-reducing end isGalNAc. Thus, it can be said that the transfer of GalNAc is performedwith respect to chondroitin having the non-reducing end of GlcUA, andthe transfer of GlcUA is performed with respect to chondroitin havingthe non-reducing end of GalNAc.

As it will be explained in the Examples below, it was confirmed that aprotein containing the amino acid sequence from amino acid numbers 47 to802 in SEQ. ID. NO: 2, has enzyme activity of human chondroitinsynthase. It was deduced that a transmembrane domain is included in theamino-acid sequence from amino acid numbers 1 to 46 in SEQ. ID. NO: 2.In this view, use of DNA not containing a sequence for encoding theamino acid sequence from amino acid numbers 1 to 46 is preferable inthat the use of such DNA enables expression of chondroitin synthase in asoluble state. More specifically, a preferable vector has “DNA encodingthe amino-acid sequence from amino-acid numbers 47 to 802, the DNAcontaining no sequence for encoding the amino-acid sequence from aminoacid numbers 1 to 46”.

In naturally-existing proteins, besides polymorphism or mutation of theDNA that codes for the protein, other mutations, such as substitution,deletion, insertion, and transposition of the amino acid in its aminoacid sequence may occur due to modification reaction of the createdprotein inside a cell or during purification. However, there has beenknown that some of the naturally-existing proteins in which suchmutation occurs, have substantially equal physiology and biologicalactivity to that of a protein in which such mutation has not occurred.The scope of the vector according to the present invention includes sucha vector having the DNA that encodes a protein that is differentslightly in terms of structure but is substantially similar in terms offunction. The same is true for a case where such a mutations isintroduced in the amino acid sequence of the protein artificially. Inthis case, it is possible to create a larger variety of mutant. Forexample, there has been known that a protein prepared by replacing, withserine, a cysteine residue in an amino acid sequence of humaninterleukin-2 (IL-2), has interleukin-2 activity (Science, 224, 1431(1984)). Further, there has been known that a kind of protein has apeptide domain that is not essential for its activity. Examples of thispeptide domain may be a signal peptide contained in an extra-cellularlysecreted protein, or a pro sequence of precursor of a protease etc. Mostof such domains are removed after translation or upon conversion into anactivated protein. Even though these proteins have different primarystructures, functions of those proteins are equivalent finally. Theforegoing DNA (b) and (c) is examples of DNA encoding such proteins.

The “stringent condition” of the DNA (b) refers to a condition forforming a specific hybrid and no unspecific hybrid. (refer to Sambrook,J. et al., Molecular Cloning A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989) etc.). The “stringent condition”may be, for example, a condition obtained by carrying out hybridizationat 42° C. in a solvent containing 50% formamide, 4×SSC, 50 mM HEPES(pH7.0), 10× Denhardt's solution, and a salmon sperm DNA of 100 μg/ml,and then washing at room temperature with 2×SSC and a 0.1% SDS solution,and sequentially washing at 50° C. with 0.1×SSC and a 0.1% SDS solution.

The “several number of amino acids” of (c) refers to an acceptablenumber of amino acids in which such mutation that does not causecatalytic activities (α) and (β) occurs. The catalytic activities (α)and (β) are explained later. For example, for a protein made of 800amino-acid residue, the acceptable number is in a range of 4 to 40,preferably 4 to 20, more preferably 4 to 10.

Note that, DNA to be carried by the vector of the present invention mayhave various nucleotide sequences due to degeneracy of genetic code.This is however easily understood by a person skilled in the art.

The catalytic activities (α) and (β) can be measured by a general assaymethod for glycosyltransferase.

More specifically, as explained in the Examples below, the catalyticactivity (α) can be measured by a method using transfer reaction ofGalNAc into chondroitin by using a UDP N-acetylgalactosamine (UDPGalNAc)as a donor, whereas the catalytic activity (β) can be measured by amethod using transfer reaction of GlcUA into chondroitin by using aUDP-glucuronic acid (UDP-GlcUA) as a donor. Accordingly, by checking thepresence of the transfer activities as an index, it is easy for a personskilled in the art to select at least one of substitution, deletion,insertion, and transposition of one or some of amino acid residues, thesubstitution, deletion, insertion, and transposition causing nosubstantial deterioration in the activity. Further, it is also possibleto easily select DNA that codes for a protein having catalyticactivities of (α) and (β) from among DNA that hybridize under the“stringent condition”.

Note that chondroitin for use herein includes both (i) one whosenon-reducing terminal is GlcUA, and (ii) one whose non-reducing terminalis GalNAc.

Moreover, the protein that the DNA (b) or the DNA (c) codes for, furtherhas the all of the following characteristics in (γ), preferably.

(γ) The following acceptors substantially receive no monosaccharide fromthe following donors (in the bracket)

-   -   Galβ1-3Galβ1-4Xyl (UDP-GlcUA)    -   GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser (UDP-GalNAc)    -   α-thrombomodulin (UDP-GalNAc)    -   sheep submandubular asialomucin (UDP-Gal)    -   GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc (UDP-Gal)

Note that, the a-thrombomodulin includes tetrasaccharide made ofGlcUAβ1-3Galβ1-3Galβ1-4Xyl. Further, the sheep submandubular asialomucincontains GalNAcα1-O-Ser/Thr.

Note that, in the foregoing condition, Gal denotes a galactose residue,Xyl denotes a xylose residue, Ser denotes a serin residue, and Thrdenotes a threonine residue, respectively. The remaining symbols are allthe same as above.

It is preferable that a protein that is coded for by the DNA carried bythe vector of the present invention is a water-soluble protein. This isbecause the water-soluble protein generally does not have atransmembrane domain, and the water-soluble protein expressed is solubleto an aqueous solvent etc., thus being easy to be purified.

The DNA carried by the vector of the present invention preferably doesnot contain DNA encoding the amino-acid sequence from amino-acid numbers1 to 46 in the SEQ. ID. NO: 2. Most preferable encodes finding fromnucleotide numbers 633 to 2900 in the SEQ. ID. NO: 1.

Further, it is further preferable that this vector is an expressionvector, since it is desirably used in the producing method ofchondroitin synthase. The method will be described later.

For example, an expression vector having DNA encoding the amino-acidsequence from amino-acid numbers 47 to 802 (DNA not containing theamino-acid sequence from amino-acid numbers 1 to 46) may be preparedwith the following method.

<A> Preparation of DNA Combined with the Vector

First, obtained is a cDNA clone (GenBank accession number AB023207)specified as “KIAA0990” in the HUGE protein database. Then, by using thecDNA clone as a template, amplification is carried out through PCRmethod with a 5′-primer (5′-CCCTCGAGGGGCTGCCGGTCCGGGC-3′ (SEQ. ID. NO:3)) containing a XhoI site, and 3′-primer(5′-CCCTCGAGCAATCTTAAAGGAGTCCTATGTA-3′ (SEQ. ID. NO:4)) containing aXhoI site 138 bp downstream from the stop codon.

The PCR method may be carried out with Pfu polymerase (Stratagene, LaJolla, Calif., USA) for 34 cycles of 94° C. for 30 seconds, 55° C. for30 seconds, and 72° C. for 180 seconds in 5% (v/v) dimethylsulfoxide.However, the PCR method may also be carried out in a general manner

<B> Introduction of DNA Fragment into Vector

The vector of the present invention may be prepared by introducing, intoa well-known vector, the DNA obtained in the foregoing manner.

The vector into which the DNA is introduced may be selected fromappropriate expression vectors(a phage vector, a plasmid vector, or thelike), which enable expression of the introduced DNA. The vector shouldenable expression of the foregoing DNA inside the host cells into whichthe vector of the present invention is transfected. Such a host-vectorsystem may be a combination of a mammalian cell such as a COS cell or3LL-HK46 cell, with an expression vector for mammalian cells such aspGIR201 (Kitagawa, H., and Paulson, J. C. (1994) J. Biol. Chem. 269,1394-1401), PEF-BOS (Mizushima, S., and Nagata, S. (1990) Nucleic AcidRes. 18, 5322), pCXN2 (Niwa, H., Yamanura, K. and Miyazaki, J. (1991)Gene 108, 193-200), pCMV-2 (Eastman Kodak), pCEV18, pME18S (Maruyama etal. Med. Immunol., 20, 27(1990)) or pSVL (Pharmacia Biotec); otherwise,a combination of a coliform (E. Coli) with en expression vector forprokaryotic cells, such as pTrcHis (produced by Inbitrogen Co., Ltd.),pGEX (produced by Pharmacia Biotec Inc.), pTrc99 (produced by PharmaciaBiotec Inc.), pKK233-3 (produced by Pharmacia Biotec Inc.), pEZZZ18(produced by Pharmacia Biotec Inc.), pCH110 (produced by PharmaciaBiotec Inc.), pET (produced by Stratagene Co.), pBAD (produced byInbitrogen Co., Ltd.), PRSET (produced by Inbitrogen Co., Ltd.), orpSE420 (produced by Inbitrogen Co., Ltd.). In addition, the host cellmay be insect cells, yeast, or grass bacillus, which are used withvarious corresponding vectors. Among these, the combination of a mammalcell and pEF-BOS is most preferable.

Further, the vector to introduce the DNA therein may be a vectorconstructed to cause expression of a fusion protein made of the proteincoded by the DNA and a marker peptide. This type of vector isparticularly preferable in the case of purifying chondroitin synthase,which is expressed by the vector of the present invention. The markerpeptide may be a protein A sequence, an insulin signal sequence, His,FLAG, CBP (calmodulin binding protein), or GST (glutathioneS-transferase), for example. Fusing of such a marker peptide to aprotein A sequence allows easy affinity purification, and fusing themarker peptide into an insulin signal sequence allows extra cellularsecretion (into culture medium etc.) of enzyme.

In the case of any vectors, the processing may be carried out in ageneral way so as to allow binding of the DNA and the vector; forexample, the DNA and the vector may be bonded together after treatingwith a restriction endonuclease or the like, and if necessary, bluntingor binding of the sticky end.

More specifically, the DNA (PCR fragment) obtained through the foregoingmethod <A> is digested by XhoI, and the both ends of the fragment arepartially filled with Klenow fragment (New England Biolabs, Beverly,Mass.), dCTP, and dTTP. Further, the pGIR201protA (J. Biol. Chem., 269,1394-1401 (1994)) vector digested by BamHI is also partially filled withdATP and dGTP. The fragments thus obtained were then subcloned into thepGIR201protA, resulting in the fusion of the DNA encoded by the DNAcreated through the method <A> with the insulin signal sequence and theprotein A sequence present in the vector. An NheI fragment containingthis fusion protein sequence was inserted into the XbaI site of theexpression vector PEF-BOS (Nucleic Acid Res., 18, 5322 (1990)), therebyobtaining an expression vector for expression of chondroitin synthasethat is fused with an insulin signal sequence and a protein A sequence.

(2) Transformant of the Present Invention

A transformant of the present invention is a transformant where a hostis transformed by the vector of the present invention (including avector containing DNA encoding a protein of amino acid sequence fromamino acid numbers 1 to 802 in SEQ ID NO. 2).

As used herein, “host” may be of any kind, provided that it can berecombined by a vector of the present invention. Preferably, the host isthe one which can make full use of the capabilities of DNA carried by avector of the present invention or a recombinant vector into which thesame DNA is recombined. Examples of the host include: animal cells;plant cells; and microorganism cells (fungus body), and COS cell(including COS-1 cell and COS-7 cell); and more specifically, amammalian cell including 3LL-HK 46 cell; a coliform (E. coli); insectcell; yeast; and grass bacillus are included, for example. The host canbe selected as appropriate in accordance with the vector of the presentinvention. However, in the case where the vector for use in the presentinvention is a vector based on PEF-BOS, for example, a cell derived froma mammal is preferably selected, and a COS cell is more preferablyselected among them.

Transformation of the host by a vector of the present invention can beperformed by standard methods known in the art. For example,transformation can be performed by introducing the vector into the hostby (i) a method using a reagent for transfection, (ii) DEAE-dextranmethod, (iii) electroporation method, or (iv) other methods.

The transformant of the present invention obtained in such a manner canbe used in applications such as production of chondroitin synthase, asdescribed later.

(3) Method of Producing Chondroitin Synthase

The method of producing chondroitin synthase of the present invention ischaracterized in that a transformant of the present invention is grownto obtain chondroitin synthase from the grown transformant.

As used herein, “growth” refers to a concept including proliferation ofa cell as a transformant of the present invention and of a microorganismitself and growth of a creature including animal and insect into which acell as a transformant of the present invention is introduced. Further,as used herein, “growth product” refers to a concept including a culturemedium after the growth of the transformant of the present invention(supernatant of a culture solution), a cultured host cell, a secretion,and an ejection.

Conditions of the growth (culture medium, culture condition, and others)are selected as appropriate in accordance with a host to be used.

According to this production method, chondroitin synthase in variousforms can be produced in accordance with a transformant to be used.

A soluble chondroitin synthase is produced by, for example, growing atransformant prepared by transformation by the expression vector havingDNA encoding the amino acid sequence from amino acid numbers 47 to 802in SEQ ID No. 2, as a vector of the present invention.

Further, an insoluble (membrane-binding) chondroitin synthase isproduced by growing a transformant prepared by transformation by theexpression vector having DNA encoding the amino acid sequence from aminoacid numbers 1 to 802 in SEQ ID No. 2, as a vector of the presentinvention.

Still further, chondroitin synthase fused with a marker peptide isproduced by growing a transformant prepared by transformation by anexpression vector constructed so as to express a fusion protein fusedwith a marker peptide.

Chondroitin synthase can be obtained from the growth product by awell-known method for protein extraction and purification, depending ona form of the produced chondroitin synthase.

For example, when chondroitin synthase is produced in a soluble formsecreted in a culture medium (supernatant of a culture solution), theobtained culture medium may be directly used as chondroitin synthase.Further, when chondroitin synthase is produced in a soluble formsecreted in a cytoplasm or in an insoluble (membrane-bound) form,extraction of chondroitin synthase can be performed by any one orcombination of a method using a nitrogen cavitation apparatus,homogenization, glass beads mill method, sonication, osmotic shock,extraction by cell homogenization using a method such as freezing andthawing method, and surface-active agent extraction. Alternatively, theextracted product may be directly used as chondroitin synthase.

It is also possible and preferable to further purify chondroitinsynthase from such culture medium and extracted product. Purificationmay be incomplete purification (partial purification) or completepurification, which may be selected as appropriate in accordance with(i) the intended use of chondroitin synthase, and (ii) the like.

Specifically, examples of purifying method include: any one orcombination of salting-out by ammonium sulphate, sodium sulphate, or thelike; centrifugal separation; dialysis; ultrafiltration; adsorptionchromatography; on-exchange chromatography; hydrophobic chromatography;reverse phase chromatography; gel filtration; gel permeationchromatography; affinity chromatography; and electrophoretic migration.

For example, when chondroitin synthase is fused with protein A toproduce a fusion protein, chondroitin synthase may be purified simply byaffinity chromatography using a solid phase combined with IgG.Similarly, when chondroitin synthase is fused with His to produce afusion protein, chondroitin synthase may be purified using a solid phasecombined with magnetic nickel. When chondroitin synthase is fused withFLAG to produce a fusion protein, chondroitin synthase may be purifiedusing a solid phase combined with anti-FLAG antibody. Still further,fusion with insulin signal eliminates the need for extracting operationsuch as cell disruption.

Production of the purified chondroitin synthase can be confirmed byanalyzing its amino acid sequence, property, substrate specificity, andothers.

(4) Reagent of the Present Invention

A reagent of the present invention is a chondroitin-synthesizingreagent, an enzyme protein having an amino acid sequence of (α) or (β),the enzyme protein having catalytic activities of (I) and (II):

(A) an amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID.NO: 2;

(B) the amino acid sequence of (A) in which one or more amino acid issubstituted, deleted, inserted, or transferred;

(α) catalytic activity that transferase GalNAc from UDP-GalNAc tochondroitin; and

(β) catalytic activity that transferase GlcUA from UDP-GlcUA tochondroitin.

The amino acid sequences of (A) and (B) are amino acid sequencesrespectively encoded by the DNA of (a) and (c), which are described inconnection with the vector of the present invention. The amino acidsequences respectively encoded by the DNA of (a) and (c) are alreadydescribed. (α) and (β) are already described in connection with thevector of the present invention.

An enzyme protein of the present invention is not limited to the enzymeprotein from amino acid numbers 47 to 802 in SEQ. ID. NO: 2. The enzymeprotein of the present invention may be, for example, an enzyme proteinfrom amino acid numbers 1 to 802 in SEQ. ID. NO: 2.

The reagent of the present invention is a chondroitin-synthesizingreagent, which makes use of effects of an enzyme protein (chondroitinsynthase) including an amino acid sequence of (A) or (B). The effectsare an effect of transferring GalNAc, and an effect of transferringGlcUA.

The reagent of the present invention is used in order to synthesizechondroitin. In the present specification, to “synthesize chondroitin′is a concept that covers extending a saccharide chain of chondroitin bytransferring and/or adding a saccharide to the chondroitin.

The reagent of the present invention is not limited to that of aparticular form. The reagent of the present invention may be in asolution form, a frozen form, or a freeze-dried form. As long as theactivities of the chondroitin synthase are not influenced, anothercomponent (e.g. a support acceptable as a reagent, or the like) may beincluded.

(5) Method of Producing Saccharide Chain

All methods of producing a saccharide chain according to the presentinvention, which use a reagent of the present invention, can becategorized into the following four types in accordance with substratesof saccharide donor and receptor used.

<1> Method of producing a saccharide chain expressed by the followingformula (3), including at least the step of bringing a reagent of thepresent invention into contact with GalNAc donor and a saccharide chainexpressed by the following formula (1)GlcUA-GalNAc-R¹   (1)GalNAc-GlcUA-GalNAc-R¹   (3)

<2> Method of producing a saccharide chain expressed by the followingformula (4), including at least the step of bringing a reagent of thepresent invention into contact with GlcUA donor and a saccharide chainexpressed by the following formula (2)GalNAc-GlcUA-R²   (2)GlcUA-GalNAc-GlcUA-R²   (4)

<3> Method of producing a saccharide chain selected from the followingformulas (5) and (7), including at least the step of bringing a reagentof the present invention into contact with GalNAc donor, GlcUA donor,and a saccharide chain expressed by the following formula (1)GlcUA-GalNAc-R¹   (1)(GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (5)GalNAc-(GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (7)

<4> Method of producing a saccharide chain selected from the followingformulas (6)and(8), including at least the step of bringing a reagent ofthe present invention into contact with GalNAc donor, GlcUA donor, and asaccharide chain expressed by the following formula (2)GalNAc-GlcUA-R²   (2)(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (6)GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (8)

As GlcUA donor, nucleotide hypophosphoric acid-GalNAc is preferable, andUDP-GalNAc is especially preferable.

As GlcUA donor, nucleotide hypophosphoric acid-GlcUA is preferable, andUDP-GlcUA is especially preferable.

The way of contacting is not especially limited provided that respectivemolecules of the chondroitin synthase, donor, and receptor (saccharidechain) are brought into contact with one another to generate enzymereaction, the chondroitin synthase, donor, and receptor being includedin a reagent of the present invention. For example, these three types ofmolecules may bring into contact with one another in a solution in whichthey are dissolved. For continuous enzyme reactions, chondroitinsynthase can be used in the form of immobilized enzyme coupled with asuitable solid phase (beads, etc.), and a membrane-type reactor using amembrane such as ultrafilter membrane and dialysis membrane may be used.As in the method described in WO 00/27437, enzyme reaction can begenerated by coupling a receptor with a solid phase. Still further, abioreactor for reproducing (synthesizing) a donor may be used together.

In the above <3> and <4> , GalNAc donor and GlcUA donor do not alwaysneed to be simultaneously brought into contact with a reagent of thepresent invention and the saccharide chain shown by the formula (1) or(2), and these donors may be alternately brought into contact with areagent of the present invention and the saccharide chain shown by theformula (1) or (2).

Although conditions for enzyme reaction are not limited provided thatchondroitin synthase acts, enzyme reaction at about neutral pH ispreferable, and enzyme reaction in a buffer having buffer action atabout neutral pH is more preferable. Further, although a temperatureduring enzyme reaction is not especially limited provided that activityof chondroitin synthase is held, about 30 to 40° C. (e.g. 37° C.) isexemplified. When there is a substance for increasing activity ofchondroitin synthase, that substance may be added. For example, it ispreferable that Mn²⁺ or other substance exists together. A reaction timecan be determined as appropriate by a person skilled in the art inaccordance with a reagent used of the present invention, the amount ofdonors and receptors, and other reaction conditions. Isolation ofchoidroitin from a reaction product, and the like process can beperformed by the well known method.

Chondroitin sulfate can be produced by using a reagent of the presentinvention (chondroitin synthase) together with sulfotransferase.

For example, in the above method of producing a saccharide chain (methodof producing chondroitin), it is possible to produce chondroitin sulfateby causing sulfate donor (3′-phosphoadenosine 5′-phosphosulfate (PAPS),or the like) to exist together with sulfotransferase to simultaneouslyperform generation of chondroitin and transfer of sulfuric acid. In thesame manner as described above, sulfotransferase may be used asimmobilized enzyme combined with a suitable solid phase (beads, etc.),and a membrane-type reactor using a membrane such as ultrafiltermembrane and dialysis membrane may be used for continuous reactions. Atthis moment, a bioreactor for reproducing (synthesizing) a sulfate donormay be used together.

Also, chondroitin can be produced by directly generating chondroitin ina host transformed by a vector of the present invention (transformant ofthe present invention).

Further, chondroitin sulfate can be directly produced in a host byintroducing a vector of the present invention and cDNA encodingsulfotransferase into a host, and causing chondroitin synthase andsulfotransferase to simultaneously express in the host (transformant ofthe present invention including cDNA encoding sulfotransferase).

Sulfotransferase (or cDNA encoding sulfotransferase) used herein may bean enzyme that transferase sulfuric acid to chondroitin (or cDNAencoding sulfotransferase) and can be selected as appropriate from thewell known enzymes in accordance with the type of a desired sulfuricacid.

Further, two or more types of sulfotransferases each having a differenttransfer position of sulfuric acid (or cDNAs encoding thesulfotransferases) may be used together.

As an example of sulfotransferase, chondroitin6-o-sulfotransferase (J.Biol. Chem., 275(28), 21075-21080 (2000)) can be given, but the presentinvention is not limited to this, and other enzyme can be also used.

(6) Probe of the Present Invention

A probe of the present invention is a probe for hybridization having anucleotide sequence from nucleotide numbers 495 to 2900, more preferably633 to 2900, in SEQ ID No: 1, or having a complementary sequencepartially in the same nucleotide sequence.

A probe of the present invention can be obtained by generatingoligonucleotide having a nucleotide sequence from nucleotide numbers 495to 2900, more preferably 633 to 2900, in SEQ ID No: 1, or having acomplementary sequence partially in the same nucleotide sequence andlabeling this oligonucleotide with a marker suitable for hybridization(e.g. radio isotope).

The length of oligonucleotide is selected as appropriate depending onconditions of hybridization using a probe of the present invention.

A probe of the present invention is expected to be a useful tool forexamining biological functions of chondroitin sulfate. This is becausechondroitin sulfate widely expresses and plays an important role in manytissues, especially in a brain. This probe is considered to be usefulfor assessment of a connection between gene and disease.

EXAMPLES

The present invention is more specifically described below withreference to examples.

Example 1

(1) In Silico Cloning of Novel Human Glycosyltransferase cDNA

Screening of HUGE protein database at Kazusa DNA Research Institute (inChiba Prefecture; http://www.kazusa.or.jp/huge/) was conducted by thekeywords “one transmembrane domain” and “galactosyltransferase family”.As a result of this, one clone (KIAA0990; GenBank™ accession numberAB023207) was identified. An analysis of a nucleotide sequence of thisclone revealed that this clone includes (i) a 5′-untranslated region of494 bp, (ii) a single open reading frame of 2406 bp coding for a proteinof 802 amino acids with three potential N-glycosylation sites (markedwith asterisks in FIG. 1), and (iii) a 31-untranslated region of about1.7 kb with a presumptive polyadenylation signal. The nucleotidesequence and an amino acid sequence deduced from the same are shown inSEQ. ID. NO: 1, whereas only the amino acid sequence is shown in SEQ.ID. NO: 2.

The clone was acquired from Kazusa DNA Research Institute. Northern blotanalysis showed that the mRNA corresponding to the clone was about 5.0kb in length in various human tissues (see Example 2), suggesting thatthe cDNA was approximately full-length. The deduced amino acid sequencecorresponded to a 91,728-Da polypeptide. A predicted translationinitiation site conformed to the Kozak consensus sequence for initiation(Nucleic Acids Res. 12, 857-872 (1984)), and an in-frame stop codonexisted upstream of an initiation ATG codon allocated thereto.

A Kyte-Doolittle hydropathy analysis (J. Mol. Biol. 157, 105-132 (1982))revealed one prominent hydrophobic segment of 17 amino acid residues inthe NH₂-terminal region, predicting that the protein has a type IItransmembrane topology which is typical in many Golgi localizedglycosyltransferases having been cloned until today (see FIG. 1).

Database searches revealed that the amino acid sequence was, at itsamino terminal, slightly homologous to human core 1 UDP-Gal:GalNAcα-Rβ1,3-Gal transferase (GenBank™ accession number AF155582), while the aminoacid sequence was, at the carboxyl terminal, slightly homologous to ahuman UDP-Gal:GlcNAcβ-Rβ1, 4-Gal transferase II (GenBank™ accessionnumber AB024434). Glycosyltransferases being homologous to the aminoacid are characterized in that the connection patterns of saccharidechains are often preserved, even though different members are specificto different donors or receptors (Biochim. Biophys. Acta 1254, 35-53(1999)).

Thus, the features of the amino acid sequence to be encoded suggested apossibility that the identified gene product could have activities ofboth the β1, 3-GlcUA transferase (GlcAT-II) and β1, 4-GalNAc transferase(GalNAcT-II). Furthermore, a homologue of the identified human gene wasfound in the Caenorhabditis elegans genome or Drosophila genome. FIG. 1illustrates that to what extent the protein sequences respectivelyderived from human, C. elegans, and Drosophila are homologous to eachother. The human protein sequence shares 36 homologies with the sequenceof the C. elegans, and 42 homologies with the sequence of theDrosophila. These three proteins all include DDD at the amino terminaland DVD at the carboxyl terminal (cf. FIG. 1), thereby being consideredas a conserved D×D motif which is found in most glycosyltransferases(Proc. Natl. Acad. Sci. U.S.A. 95, 7945-7950 (1998)).

In addition to the above, a data base search of the Human Genome Projectidentified a genome sequence (accession number NT010274.3) identicalwith the above-mentioned cDNA sequence. Comparison of the cDNA andgenome sequences revealed the genomic structure and chromosomallocalization of the gene. The gene spans over 40 kb, and the codingregion thereof was divided into three discrete exons as shown in FIG. 2.The intron/exon junctions followed the GT/AG rule and were flanked byconserved sequences. This gene is located on the human chromosome number15.

(2) Construction of a Plasmid Including DNA Encoding a Novel SolubleGlycosyltransferase

A cDNA of a Glycosyltransferase that lacked 46 amino acid residues atits N-terminal from this novel glycosyltransferase was amplified by PCR.Specifically, with a KIAA0990 cDNA as a template, amplification wascarried out using (a) a 5′-primer (5′-CCCTCGAGGGGCTGCCGGTCCGGGC-3′ (SEQ.ID. NO: 3)) including an XhoI site and (b) a 3′-primer(5′-CCCTCGAGCAATCTTAAAGGAGTCCTATGTA-3′ (SEQ. ID. NO: 4)) including aXhoI site located 138 bp downstream from the stop codon. The PCR wascarried out with Pfu polymerase (Stratagene Co., La Jolla, Calif.) for34 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 180 seconds in 5 (v/v) dimethyl sulfoxide. The PCR-product was thendigested with the XhoI. Then, at both terminals of its fragment, the PCPproduct was partially filled with Klenow Fragment (New England BiolabsInc., Beverly, Mass.), a dCTP, and a dTTP. A pGIR201protA (J. Biol.Chem. 269, 1394-1401 (1994)) vector digested with BaMHI was alsopartially filled with a dATP and a dGTP. The obtained fragment wassubcloned into the pGIR201protA, so that the novel glycosyltransferasewas fused with the insulin signal sequence and the protein A sequencewhich were carried in the vector. An NheI fragment including theabove-mentioned fusion protein sequence was inserted into the XbaI siteof the expression vector PEF-BOS (Nucleic Acids Res. 18, 5322 (1990)),whereby an expression plasmid was obtained.

This expression plasmid encodes a protein in which the first 46 aminoacids of the glycosyltransferase is replaced with a cleavable insulinsignal sequence and a protein A IgG-binding domain. In other words, theexpression plasmid encodes a soluble chondroitin synthase fused with acleavable insulin signal sequence and a protein A.

(3) Expression of a Novel Soluble Glycosyltransferase, and EnzymaticAssay Thereof

By using FuGENE (Trademark) 6 (Roche Molecular Biochemicals Co., Tokyo),an expression plasmid (6.7 μg) was transfected into a COS-1 cell on a100 mm plate, in accordance with a manual of the manufacture. On thesecond day from the transfection, 1 ml of an incubation liquid wascollected and incubated together with 10 μL of IgG-Sepharose (AmershamPharmacia Biotech) at 40C for one hour. Beads of the IgG-Sepharose wascollected by centrifugation, and then washed with an assay buffer. Afterthat, the beads were resuspended in an assay buffer of the same kind asthe assay buffer. The beads were used for assaying GalNAc transferase,GlcUA transferase, and Gal transferase. That is, a fused proteinoccurred in the incubation liquid was absorbed by IgG-Sepharose so as toremove glycosyltransferases in the incubation liquid. Then, by using theenzyme bound beads as an enzyme source, glycosyltransferase activity ofthe fused protein that was bound to the beads was assayed with variousreceptor substrates and donor substrates.

As a receptor for GalNAc transferase, a polymer (167 μg) of chondroitin,α-thrombomodulin (1 nmol), or GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser (1nmol) was used. Moreover, as a receptor for GlcUA transferase, thepolymer (167 μg) of chondroitin or Galβ1-3Galβ1-4Xylβ (1 nmol) was used.As a receptor of Gal transferase, sheep submandibular asialomucin (300μg) or GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc (1 nmol) was used. Assayof GalNAc transferase was carried out with a mixture of, in 30 μL intotal, 10 μL of the resuspended beads, the receptor substrate, 8.57 μMUDP-[³H]GalNAc (3.60×10⁵ dpm), 50 mM MES buffer, pH 6.5, 10 mM MnCl2,and 171 μM of sodium salt of ATP (J. Biochem. 117, 1083-1087 (1995)).

Assay of GlcUA transferase I (GlcAT-I), which is necessary for synthesisof tetrasaccharide for the linkage region, was carried out with amixture of, in 30 μL in total, 10 μL of the resuspended beads, 1 nmolGalβ1-3Galp1-4Xyl, 14.3 μM UDP-[¹⁴C]GlcUA (1.46×10⁵ dpm), 50 mM MESbuffer, pH6.5, and 2 mM MnCl₂ (FEBS lett. 459, 415-420 (1999)). In assayof GlcAT-II, 10 μL of the resuspended beads, 167 μL g of the polymer ofchondroitin, 14.3 μM UDP-[¹⁴C]GlcUA (1.46×10⁵ dpm), 50 mM sodium aceticacid buffer, pH 5.6, and 10 mM MnCl2 were included in 30 μL in total(Glycobiology 7, 905-911(1997)). Assay of Gal transferase was carriedout with a mixture of, in 30 μL in total, 10 μL of the resuspendedbeads, the receptor substrate, 60μM UDP-[³H]Gal (5.30×10⁵ dpm), 50 M MESbuffer, pH 6.5, 10 nM MnCl₂, and 171 μM of sodium salt of ATP. Reactionmixtures were incubated at 370C for one hour. Products that had beenradiolabeled was separated from UDP-[³H]GalNAc, UDP-[¹⁴C]GlcUA, orUDP-[³H] Gal, by gel filtration using a syringe column packed withSephadex G-25 (super fine), a superdex peptide column, or a Pasteurpipette column containing Dowex 1-X8 (PO42-type, 100-400 mesh, Bio-RadLaboratories, Tokyo) (J. Biochem. 117, 1083-1087 (1995); J. Biol. Chem.273, 6615-6618 (1998); FEBS Lett. 459, 415-420 (1999); Glycobiology 7,905-911 (1997); Glycobiology 7, 531-537 (1997)); The thus collectedlabeled products were quantified by liquid scintillation spectroscopy.

Note that the substrates and the like were obtained as follows.UDP-[U-¹⁴C]GlcUA (285.2 mCi/mmol), UDP-[³H]GalNAc (10 Ci/mmol) andUDP-[³H]Gal (15 Ci/mmol) were purchased from NEN Life Science ProductsInc. Unlabeled UDP-GlcUA, UDP-GalNAc and UDP-Gal were obtained fromSigma. Chondroitin (a derivative prepared by chemically desulfurizingchondroitin sulfuric acid A derived from whale cartilage) was purchasedfrom Sekikagaku Corp. (Tokyo). Homogeneity purified Hepatopancreasβ-glucuronidase (EC3.2.1.31) (Comp. Biochem. Physiol. 86B, 565-569(1987)) derived from Amlullaria (freshwater apple snail) was providedfrom Tokyo Internal Organ Co. Ltd. (Tokyo).

Galβ1-3Galβ1-4Xyl was kindly provided from Dr. Nancy B. Schwartz(University of Chicago). The purified α-thrombomodulin (Biochem.Biophys. Res. Coummn. 171, 729-737 (1990)) was provided from DaiichiPharmaceutical Co. Ltd (Tokyo) and included the tetrasaccharide(GIcUAβ1-3Galβ1-3Galβ1-4Xyl1) (J. Biol. Chem. 273, 33728-33734 (1998))for the linkage region. N-acetyl chondroitin (GlcUAβ1-3GalNAc) andGlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc was kindly provided from Dr. K.Yoshida (Seikagaku Corp.). Linkage tetrasaccharide-serine(GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser) (Liebigs Ann. 1239-1257 (1996)) waskindly provided from Dr. T. Ogawa (Physical and Chemical ResearchInstitute, Saitama Prefecture).

Sheep submandibular asialomucin was obtained by treating sheepsubmandibular mucin with sialidase derived from Arthrobacter ureafaciens(Nacalai Tesque Inc. Kyoto), the sheep submandubular mucin having beenprepared according to methods of Tettamanti and Pigman (Arch. Biochem.Biophys. 124, 45-50 (1968)). Superdex (Trademark) peptide HR10/30 columnwas supplied from Amersham Pharmacia Biotech (Uppsala, Sweden).

Results were shown in Table 1. Activities were detected when the polymerof chondroitin was used as the receptor and UDP-GlcUA or UDP-GlNAc wasused as the donor. On the other hand, no activity was detected when theother receptor substrate was used and one of UDP-GlcUA, UDP-GalNAc andUDP-Gal was used as the donor. Such activities included activities of(a) GlcAT-I (which relates to initiation of biosynthesis of chondroitinsulfate), (b) GalNAc transferase I, (c) core 1 UDP-Gal:GalNAc α-R β1,3-Gal transferase, and (d) UDP-Gal:GlcNAc β-R β 1,4-Gal transferase.Glycosyltransferase activity was not detected in an affinitypurification product that was a sample prepared as a control bytransfecting pEF-BOS. Those results clearly show that expressed proteinswere GlcUA/GalNAc transferases having a high specificity for the polymerof chondroitin.

As described above, chondroitin (the polymer of chondroitin) includesone whose non-reducing terminal is GlcUA and one whose non-reducingterminal is GalNAc. It can be said that the transfer of GalNAc is forthe chondroitin whose non-reducing terminal is GlcUA, and the transferof GlcUA is for the chondroitin whose non-reducing terminal is GalNAc.TABLE 1 RECEPTOR SPECIFICITY ACTIVITY 3) RECEPTOR (DONOR) (pmol/mlmedium/time) Chondroitin (UDP-GlcUA) 5.2 Galβ1-3Galβ1-4Xyl (UDP-GlcUA)ND Chondroitin (UDP-GalNAc) 1.4 GlcUAβ1-3Galβ1-3Galβ1- ND 4Xylβ-O-Ser(UDP-GalNAc) α-thrombomodulin (UDP-GalNAc) ND Sheep submandubularasialomucin ND (UDP-Gal) GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1- ND 4GlcNAc(UDP-Gal)ND: Not Detected (<0.1 pmol/ml medium/time)1) α-thrombomodulin included tetrasaccharide linkageGlcUAβ1-3Galβ1-3Galβ1-4Xyl (J. Biol. Chem. 273, 33728-33734 (1998)).2) Sheep submandibular asialomucin had a large number of GalNAc α1-O-Ser/Thr residues.3) Each value is an average of the measures taken in two independentsexperiments.(4) Identification of Enzymatic Reaction Products

Isolation of products of GalNAc transferase reaction or GlcUAtransferase reaction, in which the polymer of chondroitin was used asthe receptor, was carried out by using gel filtration using a superdexpeptide column that had been equilibrated with 0.25 mNH₄HCO₃/7-propanol. With radioactivity peak pooled, the radioactivitypeak containing each enzymatic reaction was evaporated to dryness. Thethus isolated products (about 120 μg) of GalNAc transferase reaction wasdigested, at 370C for one night, in a reaction liquid of 30 μL by using100 mIU of Chondroitinase AC-II (EC4.2.2.5) (Seikagaku Corp. (Tokyo))derived from Arthrobacter aurescens, the reaction liquid containing 50mM sodium acetic acid buffer, at pH 6.0. Degree of digestion thereof wasevaluated. The thus isolated products (about 180 μg) of GlcUAtransferase reaction was digested for one night at 370C in 30 μL of 50mM sodium acetic acid buffer, at pH6.0, containing 100 mIU ofchondroitinase AC-II, or in 30 μL of 0.05M sodium citric acid buffer, atpH4.5, containing 22 mIU of β-glucuronidase. Digestion products of eachenzyme were analyzed by using the same superdex peptide column.

An analysis result of the products of the GlcUA transferase reaction isshown in FIG. 3(a). The labeled products were completely digested byβ-glucuronidase or chondroitinase AC-II. Peaks were observed atpositions of free [¹⁴C]GlcUA or free [¹⁴C]GlcUAβ1-3GalNAc. This resultsuggests that the GlcUA residue is transferred to GalNAc that existed atthe non-reducing terminal of the polymer of chondroitin, and caused theGlcUA residue to form β1-3 bonding with GalNAc.

An analysis result of the products of the GalNAc transferase reaction isshown in FIG. 3(b). The labeled products were completely digested bychondroitinase AC-II. A Peak was observed at a position of free[³H]GalNAc. This result suggests that the GalNAc residue is transferredto GlcUA that existed at the non-reducing terminal of the polymer ofchondroitin, and caused the GalNAc residue to form β1-4 bonding withGlcUA. To sum up the results, it was found that the proteins thusidentified were chondroitin synthase that had both the activities ofGlcAT-II and GalNAcT-II.

Example 2

A commercially-available human 12-lane multiple tissue Northern blot(Clontech) membrane was used for analysis. To each lane, 1 μg of apolyadenylated RNA was applied. The membrane was probed with agel-purified and radiolabeled (>1×10⁹ cpm/μg) 0.84 kbchondroitin-synthase-specific fragment corresponding to nucleotides631-1469 of the KIAA0990 cDNA (GenBank™ accession number AB023207).

As a result, a single band of up to 5.0 kb was demonstrated for allhuman tissues, at least in this analysis (FIG. 4). The degree of theexpression of the chondroitin synthase gene which is prevalent in humantissues varied with the types of human tissues. Notably, a particularlystrong expression of the mRNA was observed in the placenta. Theexpressions observed in the spleen, lung, and peripheral bloodleukocytes were also strong but not as much as that of the placenta.This result corresponds to an observation that chondroitin sulfateproteoglycans are distributed at the surfaces of many cells and in theextracellular matrix of almost all tissues.

Industrial Applicability

Provided are (a) a vector having DNA encoding human chondroitinsynthase, (b) a method of producing human chondroitin synthase, (c) amethod of producing a saccharide chain having a repeating disaccharideunit of chondroitin, and (d) a probe for hybridization of humanchondroitin synthase.

1-15. (canceled)
 16. A vector carrying one of DNA (a), (b) or (c), the DNA (b) or (c) encoding a protein having catalytic activities (a) and (i), excluding a DNA encoding a protein at amino-acid position #1 to 802 in SEQ. ID. NO: 2): (a) DNA encoding a protein having an amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO: 2; (b) DNA that, in a stringent condition, hybridizes with the DNA (a), the DNA complementary with DNA (a),.or DNA having part of a nucleotide sequence of the DNA (a) or the DNA complementary with the DNA (a); (c) DNA encoding a protein having an amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO:2, wherein one or several amino acids in the amino acid sequence are substituted, deleted, inserted, or transpositioned; (α) catalytic activity that transferase GalNAc from UDP-GalNAc to chondroitin; (where UDP is uridine 5′diphosphate, and GalNAc is N-acetylgalactosamine residue), and (β) catalytic activity that transferase GlcUA from UDP-GlcUA to chondroitin, (where UDP is uridine 5′diphosphate and GlcUA is N-glucuronic acid residue).
 17. The vector as set forth in claim 16, wherein: the DNA (a) encodes finding from nucleotide numbers 633 to 2900 in SEQ. ID. NO:
 1. 18. The vector as set forth in claim 16, wherein the proteins are soluble.
 19. The vector as set forth in claim 17, wherein the proteins are soluble.
 20. The vector as set forth in claim 16, being an expression vector.
 21. The vector as set forth in claim 17, being an expression vector.
 22. The vector as set forth in claim 18, being an expression vector.
 23. The vector as set forth in claim 19, being an expression vector.
 24. A transformant whose host is transformed by a vector having any one of DNA (a) to (c), the DNA (b) or (c) encoding a protein having catalytic activities (a) and (f): (a) DNA encoding a protein having an amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO: 2; (b) DNA that, in a stringent condition, hybridizes with the DNA (a), the DNA complementary with DNA (a), or DNA having part of a nucleotide sequence of the DNA (a) or the DNA complementary with the DNA (a); (c) DNA encoding a protein having an amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO:2, wherein one or several amino acids in the amino acid sequence are substituted, deleted, inserted, or transpositioned; (α) catalytic activity that transferase GalNAc from UDP-GalNAc to chondroitin; (where UDP is uridine 5′diphosphate, and GalNAc is N-acetylgalactosamine residue), (β) catalytic activity that transferase from UDP-GlcUA to chondroitin, (where UDP is uridine 5′diphosphate and GlcUA is N-glucuronic acid residue).
 25. The transformant as set forth in claim 24, wherein: the DNA (a) encodes finding from nucleotide numbers 633 to 2900 in SEQ. ID. NO:
 1. 26. The transformant as set forth in claim 24, wherein: the proteins are soluble.
 27. The transformant as set forth in claim 25, wherein: the proteins are soluble.
 28. A method for producing chondroitin synthase, the method comprising the steps of: growing a transformant set forth in claim 24; and obtaining the chondroitin synthase from the transformant thus grown.
 29. A method for producing chondroitin synthase, the method comprising the steps of: growing a transformant set forth in claim 25; and obtaining the chondroitin synthase from the transformant thus grown.
 30. A method for producing chondroitin synthase, the method comprising the steps of: growing a transformant set forth claim 26; and obtaining the chondroitin synthase from the transformant thus grown.
 31. A method for producing chondroitin synthase, the method comprising the steps of: growing a transformant set forth claim 27; and obtaining the chondroitin synthase from the transformant thus grown.
 32. A reagent for use in chondroitin synthesis, the reagent having an enzyme protein that has an amino acid sequence including an amino acid sequence (A) or (B) and has catalytic activities (α) and (β) (A) amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO: 2; (B) amino acid sequence from amino acid numbers 47 to 802 in SEQ. ID. NO:2, wherein one or several amino acids in the amino acid sequence are substituted, deleted, inserted, or transpositioned. (α) catalytic activity that transferase GalNAc from UDP-GalNAc to chondroitin, (where UDP is uridine 5′diphosphate, and GalNAc is N-acetylgalactosamine residue); (β) catalytic activity that transferase GlcUA from UDP-GlcUA to chondroitin, (where UDP is uridine 5′diphosphate and GlcUA is glucuronic acid residue).
 33. The reagent as set forth in claim 32, wherein the enzyme protein is soluble.
 34. A method for producing a saccharide chain expressed by Formula (3), the method comprising at least the step of causing a reagent to contact with GalNAc donor and a saccharide chain expressed by Formula (1), the reagent set forth in claim 32: GlcUA-GalNAc-R¹   (1), GalNAc-GlcUA-GalNAc-R¹   (3), (where GlcUA and GalNAc are as defined above, “-” indicates a glycoside linkage, R¹ is an arbitrary group).
 35. A method for producing a saccharide chain expressed by Formula (3), the method comprising at least the step of causing a reagent to contact with GalNAc donor and a saccharide chain expressed by Formula (1), the reagent set forth in claim 33: GlcUA-GalNAc-R¹   (1), GalNAc-GlcUA-GalNAc-R¹   (3), (where GlcUA and GalNAc are as defined above, “-” indicates a glycoside linkage, R¹ is an arbitrary group)
 36. A method for producing a saccharide chain expressed by Formula (4), the method comprising at least the step of causing a reagent to contact with GlcUA donor and a saccharide chain expressed by Formula (2), the reagent set forth in claim 32: GalNAc-GlcUA-R²   (2), GlcUA-GalNAc-GlcUA-R²   (4), (where GlcUA, GalNAc, and “-” are as defined above, R² is an arbitrary group).
 37. A method for producing a saccharide chain expressed by Formula (4), the method comprising at least the step of causing a reagent to contact with GlcUA donor and a saccharide chain expressed by Formula (2), the reagent set forth in claim 33: GalNAc-GlcUA-R² ⁽2), GlcUA-GalNAc-GlcUA-R²   (4), (where GlcUA, GalNAc, and “-” are as defined above, R² is an arbitrary group).
 38. A method for producing a saccharide chain selected from saccharide chains expressed by Formulas (5) and (7) respectively, the method comprising at least the step of causing a reagent to contact with GalNAc donor, GlcUA donor and a saccharide chain expressed by Formula (1), the reagent set forth in claim 32: GlcUA-GalNAc-R¹   (1), (GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (5), GalNAc-(GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (7), (where n is an integer not less than 1, GlcUA, GalNAc, and “-” are as defined above, R¹ is an arbitrary group).
 39. A method for producing a saccharide chain selected from saccharide chains expressed by Formulas (5) and (7) respectively, the method comprising at least the step of causing a reagent to contact with GalNAc donor, GlcUA donor and a saccharide chain expressed by Formula (1), the reagent set forth in claim 33: GlcUA-GalNAc-R¹   (1), (GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (5), GalNAc-(GlcUA-GalNAc)n-GlcUA-GalNAc-R¹   (7), (where n is an integer not less than 1, GlcUA, GalNAc, and “-” are as defined above, R¹ is an arbitrary group).
 40. A method for producing a saccharide chain selected from saccharide chains expressed by Formulas (6) and (8) respectively, the method comprising at least the step of causing a reagent to contact with GalNAc donor, GlcUA donor and a saccharide chain expressed by Formula (2), the reagent set forth in claim 32: GalNAc-GlcUA-R²   (2), (GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (6), GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (8), (where n is an integer not less than 1, GlcUA, GalNAc, and “-” are as defined above, R² is an arbitrary group).
 41. A method for producing a saccharide chain selected from saccharide chains expressed by Formulas (6) and (8) respectively, the method comprising at least the step of causing a reagent to contact with GalNAc donor, GlcUA donor and a saccharide chain expressed by Formula (2), the reagent set forth in claim 33: GalNAc-GlcUA-R² ⁽2), (GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (6), GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (8), (where n is an integer not less than 1, GlcUA, GalNAc, and “-” are as defined above, R² is an arbitrary group).
 42. A probe for hybridization, the probe containing a nucleotide sequence from nucleotide numbers 495 to 2900 in SEQ. ID. NO: 1, or a sequence complementary with part of the nucleotide sequence. 